Gonads are dissected from 5.5 day old chicken embryos and placed in 1.5ml siliconized tubes containing 500µl of 10% P-FBS DMEM.After pooling gonads from approximately 10 embryos the samples are centrifuged at 600 x g for 5 minutes.The gonad suspension is then diluted with 200µl of a 0.25% trypsin/EDTA (Gibco, Grand Island, NY) solution and incubated for 20 minutes at 37ºC.

The gonad suspension is teased until only the homogenate remains, which is then subsequently removed.The trypsin is then deactivated by the addition of 200µl of 20% P-FBS DMEM.The cell suspension is then passed through a 30 micron filter that has been pre-wet with 50µl of 20% P-FBS DMEM and collected in a 1.5ml siliconized tube.The trypsin tube is rinsed twice with 200µl of 20% P-FBS DMEM and then passed through the filter as well.The tube is then washed twice with 200µl 0% FBS DMEM and the solutions are passed through the filter.The cell suspension is then centrifuged for 5 minutes at 600 x g.The supernatant is then removed and 500µl of 20%ES-FBS 199 is added to the cell suspension.The final cell concentration from 10 embryos should be approximately 2 X 106 cells/ml.

The cell suspension is then cooled to 5ºC over one hour and the remaining steps are performed at 5ºC.The cell suspension is centrifuged for 5 minutes at 600 x g and the supernatant is removed.The cell suspension is diluted with 150µl of 5ºC cryopreservation media, loaded into 0.5ml straws and heat sealed.The straws are frozen at a rate of -1ºC/minute to -85ºC using a programmable freezer (Mini Digitcool UJ40, Cryo Bio System, Paris, France) and then plunged into liquid nitrogen for storage.